Current cancer treatment is based mainly on chemotherapy, radiotherapy and surgery. In spite of an elevated rate of cure for cancer at early stages, most advanced cases of cancer are incurable because they cannot be extirpated surgically or because the doses of radio or chemotherapy administered are limited by their toxicity in normal cells. In order to palliate this situation, biotechnology strategies have been developed that seek to increase the potency and selectivity of oncology treatments. Among them, gene therapy and virotherapy use viruses with a therapeutic intention against cancer. In gene therapy the virus is modified to prevent its replication and to serve as vehicle or vector of therapeutic genetic material. On the contrary, virotherapy uses virus that replicate and propagate selectively in tumour cells. In virotherapy the tumour cell dies by the cytopathic effect caused by the replication of the virus in its interior rather than by the effect of a therapeutic gene. The preferential replication in a tumour cell is named oncotropism and the lysis of the tumour is named oncolysis. In a strict sense, viruses that replicate selectively in tumours are named oncolytic, although in a broader sense the oncolytic word can be applied to any replication-competent virus able to lyse tumour cells, even without selectivity. In this description the oncolytic term is used in both senses.
Virotherapy of the cancer is previous to gene therapy. The first observations of cures of tumours with viruses date from the beginning of the last century. In 1912 De Pace obtained tumour regressions after inoculating rabies virus in cervical carcinomas. Since then many types of viruses have been injected in tumours for their treatment. There are viruses that display a natural oncotropism such as autonomous parvovirus, vesicular stomatitis virus, and reovirus. Other viruses can be manipulated genetically to replicate selectively in tumours. For example, Herpes Simplex virus (HSV) has become oncotropic by eliminating the ribonucleotide reductase gene, an unnecessary enzymatic activity in cells in active proliferation such as tumour cells. However, adenovirus, due to its low pathogenicity and high capability to infect tumour cells has been the virus more often used in virotherapy and in gene therapy of cancer.
Fifty one human serotypes of adenovirus have been identified and classified in 6 different groups from A to F.
Adenovirus human type 5 (Ad5), that belongs to group C, is a virus formed by a protein icosahedral capsid that packages a linear DNA of 36 kilobases. In adults the infection with Ad5 is usually asymptomatic and in children it causes a common cold and conjunctivitis. In general Ad5 infects epithelial cells, which in the course of a natural infection are the cells of the bronchial epithelium. It enters the cell by means of the interaction of the fibre, the viral protein that extends as an antenna from the twelve vertices of the capsid, with a cellular protein involved in intercellular adhesion named Coxsackie-Adenovirus Receptor (CAR). When the viral DNA arrives at the interior of the nucleus, it begins an ordered transcription of the early genes (E1 to E4) of the virus. The first viral genes that are expressed are the genes of the early region 1A (E1A). E1A binds to the cellular protein Rb to release E2F, that activates the transcription of other viral genes such as E2, E3, and E4, and of cell genes that activate the cell cycle. On the other hand, E1B binds to p53 to activate the cell cycle and to prevent the apoptosis of the infected cell. E2 encodes proteins involved in virus replication; E3 encodes proteins that inhibit the antiviral immune response; E4 encodes for proteins involved in viral RNA transport. The expression of early genes leads to the replication of the virus DNA, and once the DNA has replicated, the major late promoter is activated and drives transcription of messenger RNA that upon differential splicing generates all the RNAs encoding for the structural proteins that form the capsid.
There are two important aspects to consider in relation to the design of oncolytic adenoviruses: selectivity and potency. In order to obtain selectivity towards the tumour cell three strategies have been used: the elimination of viral functions that are necessary for replication in normal cells but that are not needed in tumour cells; the control of the viral genes that start the replication using tumour-selective promoters; and the modification of the virus capsid proteins implied in the infection of the host cell. With these genetic modifications a considerable level of selectivity has been obtained, with a replication efficacy in tumour cells in the order of 10000 times superior to the replication efficacy in normal cells. With regard to oncolytic potency, several genetic modifications have also been described to increase it. These modifications include: a) the increase of virus release, for example by eliminating E1B19K, over-expressing E3-11.6K (ADP), or localizing E3/19K protein in the plasmatic membrane; and b) the insertion of a therapeutic gene in the genome of the oncolytic adenovirus to generate an “armed oncolytic adenovirus”. In this case, the therapeutic gene would have to mediate the death of non-infected tumour cells by means of the activation of a prodrug with bystander effect (that is to say, that kills the non-infected neighbouring cells), the activation of the immune system against the tumour, the induction of the apoptosis, the inhibition of the angiogenesis, or the elimination of the extracellular matrix, among others. In these cases, the way and the time of expression of the therapeutic gene will be critical in the final result of the therapeutic approach.
In the last decade, different oncolytic adenoviruses have been administered to patients with head and neck, ovarian, colorectal, pancreatic, and hepatocellular carcinomas, among others. The safety profile of these adenoviruses in clinical trials has been very promising. The detected adverse effects, such as flu-like symptoms and increase levels of transaminases, were well tolerated, even after the systemic administration of high doses of virus (cfr. D. Ko et al., “Development of transcriptionally regulated oncolytic adenoviruses”, Oncogene 2005, vol. 24, pp. 7763-74; and T. Reid et al., “adenoviral Intravascular agents in cancer patients: lessons from clinical trials”, Cancer Gene Therapy 2002, vol. 9, pp. 979-86). Although the administration of the recombinant adenovirus induced a partial suppression of tumour growth, the complete eradication of the tumours was not achieved and after a short period of time the tumours re-grew quickly. These results probably occurred because the injected adenovirus distributed only in a small part of the tumour to produce a limited antitumour response, as non-infected cells continued growing quickly. In a recent work, it was observed that the replication of oncolytic adenoviruses in human xenograft tumours persisted until 100 days after systemic administration, although this replication did not translate in a complete eradication of the tumour (cfr. H. Sauthoff et al., “Intratumoural spread of wild-type adenovirus is limited to after local injection of human xenograft tumours: virus persists and spreads systemically at late time points”, Human Gene Therapy 2003, vol. 14, pp. 425-33). This low antitumour efficacy is in part because the connective tissue and the extracellular matrix (ECM) in the tumour prevent the spread of adenovirus within the tumour.
This difficulty of oncolytic adenoviruses to spread efficiently within the tumour mass has been described also for other antitumour drugs such as doxorubicin, taxol, vincristine, or methotrexate. Many studies demonstrate the role of the ECM in the resistance of tumour cells to chemotherapy drugs (cfr. BP Toole et al., “Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells”, Seminars in Cancer Biology 2008, vol. 18, pp. 244-50). Tumour and stromal cells produce and assemble a matrix of collagen, proteoglycans and other molecules that difficults the transport of macromolecules inside the tumour. Hyaluronic acid (HA) is one of the main components of the ECM involved in the resistance of tumour cells to therapeutic drugs. HA is overexpressed in a great variety of malignant tissues, and in many cases the level of HA is a factor tumour progression prognosis. The interaction of HA with receptors CD44 and RHAMM increases tumour survival and invasion. In addition, HA can promote tumour metastases by inducing cell adhesion and migration, and protection against the immune system.
On the other hand, the inhibition of the interactions between hyaluronic acid and tumour cells revert the resistance to many drugs. Different studies have indicated that hyaluronidases (enzymes that degrade HA) increase the activity of different chemotherapies in patients with melanoma, Kaposi sarcoma, head and neck tumours, and liver metastases of colon carcinoma. The mechanism of action of hyaluronidases is still unknown, but generally it is attributed to reducing cell adhesion barriers, reducing interstitial pressure, and improving penetration of the antitumour drug in the tumour, rather than to its inhibitory effects of signalling pathways related to cellular survival.
Recently, it has been described that the coadministration of hyaluronidase with oncolytic adenoviruses by means of intratumoural injection, reduces tumour progression (cfr. S. Ganesh et al., “Intratumoural coadministration of hyaluronidase enzyme and oncolytic adenoviruses enhances virus potency in mestastasic tumour models”, Clin Cancer Res 2008, vol. 14, pp. 3933-41). In these studies oncolytic adenoviruses are administered in four intratumoural injections and hyaluronidase is administered intratumourally every other day during all the treatment. This regimen of administration has little application to patients because most of the tumours are inaccessible to be injected intratumourally. The patients with scattered disease (metastasis) could not benefit from the treatment proposed by Ganesh and collaborators.
In spite of the efforts to date, it is still necessary to find new therapeutic approaches effective in the treatment of the cancer.